The aim of this proposal is to understand the molecular mechanisms of cellular transformation by retroviruses. The project is divided into two main sections. The first deals with retroviruses encoding tyrosine protein kinases, a class of virus which apparently transforms cells by perturbing cellular phosphotyrosine metabolism. We plan to identify cellular proteins which are substrates for these virally-coded tyrosine protein kinases. These will be proteins containing increased levels of phosphotyrosine when isolated from transformed cells. For this purpose we will use a number of techniques including two-dimensional gel electophoresis and antibodies specific for phosphotyrosine. The substrate specificities of the virally-coded tyrosine protein kinases will be compared with each other and with the tyrosine protein kinases activated by the growth factors, epidermal growth factor and platelet derived growth factor. The goal is to find common substrates which may be important in cellular transformation and growth control. Some of these substrates will be purified in an attempt to identify their functions and to raise antisera. In the long term we hope to determine the effect of tyrosine phosphorylation on the function of these proteins. In this way we may be able to explain particular aspects of the transformed cell phenotype. The second part of the proposal concerns Moloney murine sarcoma virus (M-MuSV). This virus does not appear to transform by phosphorylating cellular proteins on tyrosine. M-MuSV therefore offers an example of another mechanism of transformation. We have recently identified the transforming protein of M-MuSV (p37mos). It is present in stable transformed cell lines in very small amounts, but at much higher levels in acutely infected cells. This suggests both that p37mos may be toxic at high concentrations and that it may possess an enzymatic activity. We plan to purify p37mos and determine its intracellular location. The purified protein will be tested for a variety of activities. We hope to idenfity the function of p37mos in order to understand its role in malignant transformation.